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. Author manuscript; available in PMC: 2018 Jun 15.
Published in final edited form as: Nephron. 2017 Jun 15;137(1):68–76. doi: 10.1159/000456016

Expression of TLR2, NOD1 and NOD2 and the NLRP3 inflammasome in renal tubular epithelial cells of male versus female mice

Sean E DeWolf 1, Alana A Shigeoka 1, Andrew Scheinok 1, Sashi G Kasimsetty, Alexander K Welch, Dianne B McKay 1,2
PMCID: PMC5599100  NIHMSID: NIHMS871967  PMID: 28614830

Abstract

Background

Sex biased outcomes are associated with acute kidney injury (AKI) and human and animal studies have shown females are preferentially protected from renal ischemia. Why females are protected is not known. One clue might lie with pattern recognition receptors (PRRs), which are triggers of ischemic injury when ligated by molecules in the ischemic milieu. Several PRR families are expressed by renal tubular epithelial cells (RTEs) and incite cell death signaling and production of pro-inflammatory molecules. Blockade of specific PRRs (e.g., TLR2, NOD1, NOD2 and NLRP3) provides highly significant protection from ischemic RTE injury. We tested whether constitutive sex-based differences exist in expression of these PRRS in RTEs, as a first step to understand sex-biased outcomes of AKI.

Methods

To determine whether PRR expression differences exist, primary RTEs isolated from male and female WT kidneys were examined by FACS, qPCR and Western Blot for expression of TLR2, NOD1, NOD2, and NLRP3 inflammasome components.

Results

No RTE sex-based differences in TLR2, NOD1, NOD2, NLRP3 or ASC were found. RTEs from female kidneys had approximately half the mRNA, but the same protein, concentration of pro-caspase-1 compared to RTEs isolated from male kidneys.

Conclusions

Our findings indicate intrinsic sex differences in RTE expression of TLR2, NOD1, NOD2, NLRP3, and ASC are not responsible for the sex-biased outcomes observed in IRI. The lower caspase-1 mRNA expression in RTEs from females warrants further exploration of additional upstream signals that might differentially regulate caspase-1 in male versus female RTEs.

Keywords: Acute kidney injury, NOD-like receptors, pattern recognition receptors, toll-like receptors, sex differences

Introduction

AKI occurs commonly and is a major cause of morbidity and mortality in hospitalized patients [1,2]. Ischemic renal injury is studied in experimental models of ischemia/reperfusion injury (IRI), where it has been shown that renal tubule cells located in regions of the kidney most susceptible to low blood flow experience the greatest injury [3,4]. Recent data have shown that after an ischemic insult renal tubular epithelial cells (RTEs) release their intracellular contents, containing molecules with structural motifs recognized by PRRs. Ligation of these receptors results in the production of pro-inflammatory cytokines and/or the induction of cell death signaling, further propagating injury to the kidney [5].

It is well known that female mice are resistant to IRI and, in fact, many experimental models exclude female mice in IRI research [6-8]. The sex-specific effect demonstrated in experimental models is paralleled by observations from human trials showing that outcomes for men are worse than for females after AKI; and males are more likely to develop CKD after AKI [9,10].

The mechanisms behind the sex differences in susceptibility to renal IRI are not yet known, and many investigative studies to date have focused on the role that sex hormones play in renal injury. While conflicting reports exist, there is evidence to suggest that estrogen has protective effects while androgens seem to worsen IRI [6,11,12].

Data from our lab and others have shown that mice with targeted deletions in TLR2, the NLRP3 inflammasome, and the cytosolic NOD2 receptor are highly protected from renal IRI [13-16]. This protection appears to be specific to the renal tubule epithelium, as WT mice transplanted with bone marrow deficient in these PRRs experience the same injury as mice with WT bone marrow cells [14]. Deletions in other PRRs (e.g., TLR4) have also been associated with protection from renal AKI, and they exert additional effects on renal endothelium and leukocytes [17,18].

The National Institutes of Health has recently recognized the importance of considering sex in experimental study design and now requires sex and gender inclusion plans in preclinical research [19]. Understanding the differences or similarities in expression of TLRs and NLRs in RTEs of males versus females is an area of IRI research that has not previously been addressed. In this study we ask whether there are intrinsic differences in baseline or stimulated expression of TLR2, NOD1, NOD2 and the NLRP3 inflammasome; PRRs that we have found to have a primary effect on renal tubular epithelial cells in renal IRI. Studying the RTEs isolated from male versus female kidneys provided a first step to understanding how sex influences renal responses to ischemia.

Methods

Mice and Isolation of renal tubular epithelial cells

Female and male C57BL/6 mice were purchased from Jackson Laboratories and housed at UCSD vivarium. Handling and use of animals was in accordance with the standards of the Association for the Assessment and Accreditation of Laboratory Animal Care. All mice used for experiments were age 6 to 8 weeks of age. RTEs were isolated from the murine kidneys and cultured ex vivo, as has been previously described [14,15]. RTEs were analyzed at approximately 5 days post-isolation, when cells had reached approximately 70% confluency.

Stimulation of renal tubular epithelial cells

Stimulation for qPCR

Cells were cultured on collagen plates in a monolayer then washed twice with 3mL of DMEM:F12 (Life Technologies, Carlsbad, CA), trypsinized and 4×10(x002C6)5 cells stimulated with either 1μg/mL of Pam3Cys (Alexis Biochemical, Farmingdale, NY) dissolved in DMEM:F12 or DMEM:F12 alone and placed in a water bath at 37°C for 3 hours with gentle agitation every 20 minutes. After 3 hours the cells were pelleted at 1200 RPM for 5 minutes, media removed and washed with 1mL of sterile PBS (Life Technologies, Carlsbad, CA), and cell pellets stored at -80°C until RNA isolation could be performed

Stimulation for western blot

The stimulation procedure was identical to above, except for the following changes: 1) 5×10(x002C6)5 cells were added to each tube; 2) cells were stimulated for 1 hour with 10 μg/mL of Pam3Cys; 3) cells were washed with protease inhibitor-containing wash buffer instead of PBS. The wash buffer contained 0.4mM Sodium-Orthovandate+5mM NaF+4.46mg/mL sodium pyrophosphate (Sigma, St. Louis, MO) in PBS.

FACS analysis

Renal tubular epithelial cells were grown in collagen plates as indicated above. Cells were trypsinized and counted. 1×10(x002C6)6 were added to FACS tubes and spun down at 1200 RPM for 5 minutes. Media was removed and cells resuspended in 400μL of FACS wash buffer. Cells were then divided evenly into individual wells of a Costar round bottom 96-well plate (Corning, Corning, NY) and stained with TruStain FcX (Biolegend; San Diego, CA) in FACS wash buffer at 1:50 dilution and placed on ice for 5 minute incubation. 50μL of PE-conjugated anti-TLR2 (eBiosciences; San Diego, CA) in FACS wash buffer (1:50 dilution) was then added to cell suspensions (final dilution of 1:100) and placed on ice away from light for 45 minutes incubation. Stained cells were washed two times with FACS wash buffer and placed in 1.2mL micro titer tubes on ice (Fischer Scientific; Hampton, NH) for FACS analysis. Cells were run on the BD Biosciences LSRIII and analysis performed on FlowJo software.

Western Blot

Equal amounts of protein were added to each well along with 3μL of TNF-treated and TNF-non treated cell extracts (Cell Signaling, Danvers, MA, #9243 and #9243s respectively) to act as positive and negative controls. Blots were blocked over night with 5% milk powder in TBST. Primary antibody staining was performed over 2 hours while secondary antibody staining was performed over 1 hour. Antibodies for protein detection of NLRP3, ASC, pro-caspase-1, pro-IL-1b, NOD1, NOD2, RIP2 proteins were purchased from the following sources with listed catalogue numbers: NLRP3 (R&D systems, Minneapolis, MN, # MAB7578); ASC (ENZO Life Sciences, Farmingdale, NY, # ADI-905-173-100); pro-caspase-1 (EMD Millipore, Temecula, CA, #EMD06-503); pro-IL-1b (Cell Signaling, # 12426); NOD1 (Cell Signaling, # 12426); NOD2 (Santa Cruz, Dallas, Tx, #sc56168; RIP2 (Cell Signaling, #4142). Both pNF-kB (Ser536) and Ikβα (L35A5) antibodies were purchased from Cell Signaling (#3033s and #4814 respectively). Blots were analyzed using Syngene PXi gel imager (Syngene, Frederick, MD) and manual band quantification performed on GeneSys Gene Tools software.

RNA, cDNA isolation and qPCR

RNA was isolated using the Zymo Research Quick-RNA MiniPrep (Zymo research corp, Irvine, CA) according to the manufacturer's instructions. cDNA was isolated using the Invitrogen First-Strand cDNA synthesis kit according to the manufacturer's instructions. All primers for the qPCRs were purchased from Genecopoeia Biotechnology Company (Rockville, MD) or Qiagen (Hilden, Germany). cDNA was standardized to 300ng/μL and then 2μL of cDNA was added to 4μL of master mix containing 1μL primer:3μL SSO Advanced Universal SYBRgreen (Bio-Rad, Irvine, CA). qPCR was performed on the Illumina Eco Real-Time PCR system with GAPDH as the housekeeping gene. In order to calculate relative expression at baseline, the ΔCq values for the male no ligand treatment were first averaged. This average was then considered the reference to which all of the female mice were compared. ΔΔ Cq values were then calculated by subtracting ΔCq [female]- ΔCq [male average]. This process was also performed with females as the reference, and there was no difference in the results. Calculations for relative expression were as follows:

Baseline expression TLR2 stimulation
Relative expression = 2ΔΔCq Relative expression=2ΔΔCq
ΔΔCq = ΔCq(female)- ΔCq(male avg) ΔΔCq = ΔCq(Pam3Cys)- ΔCq(no ligand)
ΔCq = Cq(gene of interest)-Cq(GAPDH) ΔCq = Cq(gene of interest)-Cq(GAPDH)
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Statistics

The numbers of male and female mice used for each experiment are described in the figure legends. Relative expression of mRNA was compared using a two tailed student's t test (GraphPad Software, Inc. La Jolla, CA), with p<0.05 considered significant and >0.05 indicating no difference between the two groups.

Results

Female mice are resistant to renal IRI and therefore models of AKI often employ male mice [6-8]. These observations prompted us to ask whether there were constitutive differences in PRR expression between male versus female RTEs.

Baseline PRR expression

Cell surface expression of TLR2 is highest in the areas of the kidney that are most susceptible to ischemic injury, primarily the proximal tubular epithelial cells located in the outer medulla and cortex of the kidney [13]. Blockade of TLR2 has been shown to prevent AKI and prevent renal tubular apoptosis and necrosis in murine models of IRI [13,20-22]. As shown in Figure 1 Panel A, we found equivalent baseline TLR2 cell surface expression on primary RTEs isolated from male versus female kidneys (Figure 1, Panel A, male- dotted line; female - solid line; and Panel B, male - black square; and female - grey square).

Figure 1.

Figure 1

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Baseline expression of TLR2 on primary unstimulated RTEs isolated from male versus female mice. Panel A. Histogram showing cell surface expression of TLR2 on primary unstimulated RTEs isolated from male versus female mice (dotted lines, male; solid line female). The data represents 1 of 3 identical experiments comparing RTEs from 3 males and 3 females per group. Panel B. Baseline mRNA expression of TLR2 comparing primary unstimulated RTEs isolated from male versus female mice. Male mice were the standard to which females were compared (relative expression). Error bars represent standard deviation (n=6 males and 5 females). No significant differences were detected between the groups. The data represents 6 identical experiments.

We next asked whether other PRRs shown to be important in renal tubules after IRI, were differentially expressed in male versus female RTE cells. Work from our lab and others has shown blockade of the NLRP3 inflammasome provides highly significant protection from renal IRI, and that production of pro-inflammatory cytokines IL-1b and IL-18 in RTEs decreases significantly in the absence of NLRP3 [15,23]. To evaluate expression of the NLRP3 inflammasome, we examined the relative expression of NLRP3, its co-receptor molecule ASC, and its downstream targets pro-caspase-1, pro-IL-1b and pro-IL-18 mRNA in resting RTE cells from male versus female mice (Figure 2, males –black square; females – grey square). Shown in Figure 2, Panel A, no differences were detected in baseline mRNA expression of NLRP3, ASC, pro-IL-1b nor pro-IL-18 between female and male mice. There was however a statistically significant difference in expression of pro-caspase-1 mRNA in RTEs between males and females with females having half the expression of pro-caspase-1 as males (Figure 2, Panel A, caspase-1). Confirming the mRNA expression, we also found that there were no differences in the protein expression of NLRP3, ASC, or pro-IL-1b in lysates of RTEs from male versus female mice and interestingly there was equivalent protein expression of pro-capase-1 in male and female RTEs (Figure 2, Panel B).

Figure 2.

Figure 2

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Baseline expression of NLRP3 inflammasome components on primary unstimulated RTEs. Panel A. Baseline mRNA expression of NLRP3 inflammasome components NLRP3, ASC, pro-caspase-1 and downstream targets pro-IL-1b and pro-IL-18. Unstimulated primary RTEs isolated from male versus female mice were compared (black box male, grey box female). Male mice were the standard to which females were compared (relative expression). The data represents 6 identical experiments. Error bars represent standard error of the mean. No statistical differences were detected, except in pro-caspase-1 (p=0.02). Panel B. Baseline protein expression of NLRP3 inflammasome components NLRP3, ASC, pro-caspase-1, pro-IL-1b. The corresponding densitometry is shown in a bar graph accompanying the Western Blots. No statistical differences were detected. The blot represents 6 identical experiments.

To further evaluate PRRs found to be important in renal IRI, we next investigated baseline mRNA expression of the best-characterized cytosolic NLRs, NOD1 and NOD2. As was the case with the inflammasome components, we found no differences in constitutive NOD1 or NOD2 expression between male and female RTE cells (Figure 3, Panel A, NOD1, NOD2; male – black square; female – grey square). We also analyzed for baseline mRNA expression of RIP2, a downstream NOD signaling mediator [24-26], and found no significant differences between males and females (Figure 3, Panel A, RIP2, male – black square; female – grey square). There was also no difference between baseline IL-6 expression between male versus female RTEs (Figure 3, Panel A, IL-6, male – black square; female – grey square). We also found no differences in protein expression of NOD1, NOD2 or RIP2 between male and female mice (Figure 3, Panel B).

Figure 3.

Figure 3

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Baseline mRNA expression of NOD1, NOD2, RIP2 and IL-6. Panel A. Unstimulated primary RTEs isolated from male versus female mice were compared (black box male, grey box female). Male mice were the standard to which females were compared. In each group 6 males were compared to 6 females. Error bars represent standard error of the mean. No statistical differences were detected between male versus female groups. Panel B. Baseline protein expression of NOD1, NOD2, and RIP2. The corresponding densitometry is shown in a bar graph accompanying the Western Blots. No statistical differences were detected between male versus female groups. The blot represents 6 identical experiments.

Upregulation of inflammasome components in RTEs after stimulation

Stimulation of TLR2 has been shown to upregulate several cytoplasmic PRRs, including NLRP3 as well as its substrates pro-IL-1b and pro-IL-18 [27-29]. To determine whether NLRP3 inflammasome components, or downstream targets pro-IL-1b and pro-IL-18, were differentially regulated between male versus female RTEs, we stimulated RTE cells with the ultrapure synthetic ligand for TLR2, Pam3Cys. We found that pro-IL-1b and NLRP3 were upregulated rapidly after Pam3Cys exposure, as has been previously reported [28]. There was a trend towards lower expression of pro-IL-1b in female mice, but it did not reach statistical significance, and no significant difference was seen in TLR2-induced NLRP3 expression in RTEs from male versus female kidneys (Figure 4, Panel A, pro-IL-1b, Nlrp3, male – black square; female – grey square). We also analyzed ASC, pro-caspase-1 and pro-IL-18 mRNA expression, and found no TLR2-induced differences between male versus female RTEs (Figure 4, Panel A, male – black square; female – grey square).

Figure 4.

Figure 4

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TLR2 induced expression of the cytoplasmic innate immune receptors NLRP3 and NOD1/NOD2 family members. Panel A. TLR2-induced mRNA expression of NLRP3 inflammasome components (NLRP3 and ASC, caspase-1) and the downstream targets IL-b, IL-18. Primary RTEs from male (black box) versus female (grey box) mice were compared after anti-TLR2 stimulation. Panel B. TLR2-induced mRNA expression of NOD1, NOD2, RIP2 and IL-6 in primary RTEs isolated from male (black box) versus female (grey box), after anti-TLR2 stimulation. In both panels error bars represent standard error of the mean. No statistical differences were detected between male versus female groups. The data represents 6 identical experiments.

Upregulation of NLRs, RIP2 and IL-6 in response to TLR2 stimulation

Significant cross talk exists between TLR2 and NOD1 and NOD2 signaling pathways [24,25,30], which prompted us to ask whether TLR2 stimulation induced differential upregulation of these NLRs in RTEs from male versus female mice. NOD2 was upregulated by 2.5 fold after TLR2 stimulation, but there were no significant differences in the induced expression in RTEs between the two groups (Figure 4, Panel B, NOD1, NOD2; male – black square; female – grey square). NOD1 mRNA expression was not induced by TLR2 stimulation in either males or females. RIP2 has been shown to be upregulated by TLR2 in other organs [31,32]. Interestingly we found that RIP2 was significantly upregulated by TLR2 stimulation in murine RTEs, which is the first time to our knowledge that TLR2-induced RIP2 expression has been shown in RTE cells (Figure 4, Panel B, IL-6; male – black square; female – grey square). There was however no difference in RIP2 expression between TLR2 stimulated RTEs isolated from male versus female mice. IL-6 mRNA was also upregulated by TLR2 activation, and again there were no differences between male versus female RTEs (Figure 4, Panel B, IL-6, male – black square; female – grey square).

TLR2 activation induces downstream NF-kB transcription [33] and we asked whether stimulation with Pam3Cys produced differences in NF-kB activation in RTEs from male versus female kidneys. As shown in Figure 5, there was no appreciable difference between TLR2-induced NF-kB activation in male versus female RTEs (Figure 5, Panel A, pNF-kB, IkBa and Panel B male – black square; female – grey square)

Figure 5.

Figure 5

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TLR2 induced activation of NF-kB in primary RTEs from male versus female mice. Panel A. Western blot of pNF-kb and its inhibitory regulator Ikβα after TLR2 stimulation, comparing primary male versus female RTEs. The data represents one of two identical experiments with one male and one female per group. Panel B. Band quantification ratio of pNF-kB to its inhibitor Ikβα. Higher values indicate greater activation. No stimulation (-), Stimulation with the TLR2 ligand PAM3CSK (+). TNF treated extracts are shown as controls TNF treated (+) or untreated (-) cell extract.

Discussion

Our study found no sex-biased differences in baseline or induced expression of TLR2, NOD1, NOD2 or NLRP3 inflammasome components in RTEs isolated from male versus female kidneys. These data suggest that differences in expression of these PRRs do not account for the natural protection afforded female mice from ischemic renal injury.

Renal ischemia reperfusion injury causes significant morbidity and mortality in hospitalized patients and contributes to delayed graft function in transplant recipients [1,2,34]. Female sex is protective for IRI in both humans and animal models, and therefore female mice are generally excluded from experimental models that are used to study ischemic renal injury [6-8]. The mechanisms behind the renal protection afforded by the female sex are not known. One hypothesis is that sex hormones confer protection, and indeed many studies to date have focused on the role of sex hormones in renal protection. In studies of organ ischemia, estrogen has been shown to have protective effects [11,12,35,36], while androgens worsen injury [6,11,12]. However, contradictory data exists even within studies, highlighting the complex nature of a potential hormonal influence.

An alternative hypothesis is that males and females have intrinsic differences in the expression of intracellular signaling molecules that are important for injury responses in the kidney. Arnold et al. showed that cardiac IRI was worsened in mice with two X chromosomes as compared to those with one X chromosome, irrespective of which sex organs were present, indicating that IRI can be influenced by genetic factors unrelated to hormones [37]. This observation is contrary to the observation that renal IRI is worse in males, however it highlights the importance of considering intrinsic, non-hormonal differences between males and females.

In the current paper we ask whether intrinsic differences exist in TLR2, NOD1, NOD2 or NLRP3 inflammasome components between RTEs isolated from male versus female kidneys. PRRs are constitutively present on RTEs and are ligated by molecules released during renal ischemia, thereby propagating renal injury after an ischemic insult by causing upregulation of inflammatory cytokines and by activating cell death signaling pathways [5,25,38,39]. Knockout models have shown that mice lacking specific PRRs, such as TLR2, the NLRP3 inflammasome and NOD2 experience significant renal protection from IRI [13-15,40-42], however mice defective in NLRP3 are also protected from rhabdomyolysis-induced AKI [43], but not from cisplatin-induced acute kidney injury [42]. A reasonable question to ask is whether males have higher expression levels of these molecules in their kidneys or whether they upregulate them more robustly, resulting in a sex-biased predisposition to renal IRI. Many studies have shown that sex hormones influence PRR expression in inflammatory cells. For instance, Jitprasertwong et al showed that TLR2 is downregulated in monocytes by both β-estradiol and progesterone [44]. Specific TLR transcripts in platelets have been found to be more abundant in women and to have distinct associations with cardiovascular risk and inflammatory biomarkers that vary by sex [45]. Coxsackie B infection causes an up-regulation of TLR2 in female (and TLR4 in male) splenic lymphocytes and this differential expression contributes to the disease resistance in females and disease susceptibility in males [46]. Sex-dependent differences have also been shown in the NLRP3 and AIM2 inflammasomes; NLRP3 is hyperactivated in macrophages of both male and female systemic lupus erythematosis patients, but the mechanisms underlying NLRP3 hyperactivation might be different between the sexes, and it was postulated that the AIM2 inflammasome might also contribute to the female sex bias of SLE pathogenesis and severity [47].

The conclusion of this study was that no differences were detected in TLR2 cell surface expression, NLPR3 inflammasome components or NOD1 and NOD2 cytosolic expression in male versus female RTEs. These findings suggest that intrinsic differences in RTE expression of these PRRs between males and females are not responsible for the sex-biased outcomes observed in IRI. We did however find that RTEs from female kidneys had about half the mRNA expression of pro-caspase-1 than those from male kidneys. This is intriguing because caspase-1 inhibition has been known for some time to have a protective effect on hypoxic injury of proximal tubular cells in rodent models [48] and caspase-1-/- mice are protected from both cisplatin-induced AKI and ischemic AKI [49,50]. Our data showed no difference in pro-caspase-1 protein between male and female RTEs, suggesting that additional studies are needed to sort out the reason for the differential expression between the mRNA and the protein forms of pro-caspase-1 in male versus female RTEs. Pro-caspase-1 is cleaved to its active form by the NLRP1, NLRC4 or AIM2 inflammasomes [51] and so future studies will also examine for the role of these inflammasomes, or their interacting proteins (e.g. NLRP3/MAVs [52]), in male versus female caspase-1 expression. Future studies will also be conducted to explore the role of sex hormones on PRR expression.

Acknowledgments

The work presented in this manuscript was supported by grants awarded to DBM from the NIH R01 DK091136, R01 DK075718 and CIRM RB5-07379.

Footnotes

Conflicts of interest/declaration of transparency: The authors have no conflicts of interest to report that have relevance to the data reported in this manuscript.

Ethical approval: All procedures performed in studies involving animals were in accordance with the ethical standards of the University of California San Diego and were conducted under approval from the Institutional Animal Care and Use Committee (IACUC) approval (IACUC approval number S12261). This article does not contain any studies with human participants performed by any of the authors.

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